Compositions and products containing S-equol, and methods for their making

ABSTRACT

A composition for use in making commercial food and skin products comprising S-equol or mixtures, including both a non-racemic mixture and a racemic mixture, of S-equol and R-equol. The composition can be used to make articles of commerce such as food supplements, pharmaceuticals, and medicaments. The compositions are useful in a method of delivering S-equol to a mammal to prevent or treat a disease or associated condition, including hormone-dependent diseases or conditions such as cardiovascular disease, lipid disorder, osteopenia, osteoporosis, liver disease, and acute ovarian estrogen deficiency. The S-equol enantiomer can be produced in a biological synthesis from the metabolism of an isoflavone by an organism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/625,934, filed Jul. 24, 2003, which claims benefit of U.S.Provisional Application No. 60/398,270, filed Jul. 24, 2002, thedisclosures of which are hereby incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

The nutritional value of soybeans and foods made of purified soyproteins is well established and the renaissance of interest in soyfoods is largely the result of documented research of the potentialhealth benefits of isoflavones, a class of phytoestrogens found inabundance in soybeans. Although the recent FDA approval allowingmanufacturers of soy foods to make a heart health claim for soy foodscontaining the mandatory 6.25 g/serving of soy protein (FDA, 1999) didnot recognize the value of soy's constituent isoflavones, studies nowindicate that phytoestrogens contribute to the cholesterol-loweringeffect while also having important non-steroidal properties thatcontribute to reduced cardiovascular risk factors. The low incidence ofhormone-dependent diseases in Asian countries where soy is consumedregularly has been suggested to be due in part to the actions of soyisoflavones.

Phytoestrogens, particularly the isoflavones derived from soy, cloverand kudzu, such as genistein, daidzein, glycitein, peurarin, and theirglycosidic derivatives, biochanin A and formononetin, and theirglycosidic derivatives, exhibit estrogenic properties in some mammalianand human tissues, and exhibit anti-estrogenic properties in othertissues by competitively inhibiting estrogen binding at estrogenreceptor sites. Unlike estrogens, these isoflavone phytoestrogens seemnot to be associated with an increased risk of breast and uterinecancers, and may actually inhibit the development of breast and prostatecancers.

Cardiovascular disease is a leading cause of morbidity and mortality,particularly in the United States and in Western European countries.Several causative factors are implicated in the development ofcardiovascular disease including hereditary predisposition to thedisease, gender, lifestyle factors such as smoking and diet, age,hypertension, and hyperlipidemia, including hypercholesterolemia.Several of these factors, particularly hyperlipidemia andhypercholesterolemia, contribute to the development of atherosclerosis,a primary cause of vascular and heart disease.

A high blood cholesterol concentration is one of the key risk factorsfor vascular disease and coronary heart disease in humans. Elevated lowdensity lipoprotein cholesterol (hereafter “LDL-cholesterol”) and totalcholesterol are directly related to an increased risk of coronary heartdisease [Cholesterol and Mortality: 30 Years of Follow-Up from theFramingham Study, Anderson, Castelli, & Levy, JAMA, Vol. 257, pp.2176-80 (1987)] while a low level of high density lipoproteincholesterol (hereafter “HDL-cholesterol”) is also a predisposing factor.Several clinical trials support a protective role of HDL-cholesterolagainst atherosclerosis. A study has shown that for every 1-mg/dLincrease in HDL-cholesterol in the blood, the risk for coronary vasculardisease is decreased by 3% in women [High-density LipoproteinCholesterol and Cardiovascular Disease: Four Prospective AmericanStudies, Gordon, Probstfield, and Garrison et al., Circulation, Vol. 79,pp. 8-15 (1989)].

Estrogens play an important role in regulating lipid metabolism andmaintaining healthy blood vessels, as evidenced by the escalation inplasma cholesterol that occurs aftermenopause and the fact thatcardiovascular disease kills more women than men in the USA and mostWestern countries. For this reason, there has been a long held beliefthat HRT would benefit postmenopausal women by providing protectionagainst CVD. The recent findings from the Women's Health InitiativeStudy of over 16,608 postmenopausal women taking HRT over an eight-yearperiod has failed to show such benefits, and actually found an increasedrisk of death from thromboembolism and heart disease, especially in thefirst year of taking combined estrogen and progestin regimen, whilesignificantly increasing the risk of breast cancer. As a consequence ofthese reports, HRT use has plummeted and women are now increasinglyseeking alternative forms of estrogen to provide the benefits ofpostmenopausal estrogen deficiency. Phytoestrogens, such as isoflavonesthat act as natural selective estrogen receptor modulators by virtue ofthe conformational binding to the estrogen receptor are potentialattractive alternatives and while there has been much published on theuse of soy or clover isoflavones, there is a paucity of data on thepotential value of the important metabolite, equol.

Recent studies have determined that soy isoflavones play a role inlowering blood concentrations of total cholesterol and LDL-cholesterolin animals, inhibiting the development of atherosclerosis. The effect ofisoflavones on blood cholesterol levels in humans is more controversial,but several studies now show the need to have isoflavones present in soyprotein to observe cholesterol-lowering effects. A key study by Crouseet al, showed a dose-dependent relationship between the reduction inserum total and LDL-cholesterol and the amount of isoflavones present insoy protein. Independent of the effects isoflavones may have oncholesterol homeostasis, there is now evidence that isoflavones exertimportant effects on blood vessels. Studies have shown reductions inlipid peroxidation, improvements in arterial reactivity, blood flow, andblood pressure, and decreases in platelet aggregation. We have recentlyfound that a daily diet containing isoflavones reduced the level ofC-reactive protein, which is one of the key markers of inflammation, andconsidered one of the precipitating factors in cardiovascular disease.All of the above are crucial risk-reduction factors for cardiovasculardisease.

Isoflavones have been shown to have bone-sparing effects. Thus far 17 invitro studies of cultured bone cells, 24 in vivo studies of animalmodels of postmenopausal osteoporosis, and 17 dietary interventionstudies show that isoflavones have bone-sparing effects. In all of thesestudies it has been the soy isoflavones or clover isoflavones that havebeen examined. We have shown for the first time that equol is animportant bone-trophic agent and that unlike estrogens, it has theability to not only reduce the activity of the bone-resorbing cells, butcan actually increase bone mineral density in postmenopausal women.

While the bulk of the scientific literature has focused on the naturalisoflavones in soy or clover, little has been reported on the actions oreffects of their intestinally derived metabolites and there remains aneed to develop further compounds and methods that can safely providetreatment or preventive benefits in mammals and humans.

Equol (7-hydroxy-3-(4′-hydroxyphenyl)-chroman), a non-steroidalestrogen, was first isolated and identified from pregnant mares urine in1932 and was later identified in the urine of humans consuming soy food.Equol has a structure similar to the steroidal estrogen estradiol. Equolis unique among the isoflavones in that it possesses a chiral center andas such exists as two distinct enantiomeric forms, the R- andS-enantiomers. All previous studies on equol appear to have beenconducted with the racemic form of equol. There has in general been alack of appreciation that two forms of equol exist and to our knowledgeno previous study has reported on the specific actions or activity ofthe individual enantiomers. Equol when originally identified in mare'surine was reported to be optically active, existing as the R-enantiomer.Later, this was found to be an incorrect assignment and evidence wasprovided that the form of equol isolated from horse urine was in factthe S-enantiomer. For the first time, we have evidence that the humanform of equol produced in the intestine, is exclusively theS-enantiomer, and we have synthesized and isolated the individualenantiomers, and shown significant differences in their respectiveaffinities for estrogens receptors (ER), ERα and ERβ.

While equol was originally found to have no estrogenic action wheninjected into ovariectomized mice in large doses, later findings showedthat it was the agent responsible for an infertility syndrome in sheep.

Also, (−)equol was originally reported as having no estrogenic activityin the ovariectomized mouse, but later the racemic mixture of equolproved to behave as a weak estrogen, while its precursors, daidzein andformononetin had no or negligible estrogenic activity.

Equol is not normally present in the urine of most healthy adults unlesssoy is consumed. The formation of equol in vivo has been exclusivelydependent on intestinal microflora as evidenced from the finding thatgerm-free animals do not excrete equol, and that equol is not found inthe plasma and urine of newborn infants fed exclusively soy foods frombirth.

Equol is exclusively a non-steroidal estrogen that does not occurnaturally in any plant-based products.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a composition for use in makingcommercial products, comprising S-equol.

The invention further relates to an article of commerce comprising anon-racemic mixture of S-equol and R-equol.

The invention further relates to a food composition comprising anadditive component comprising S-equol.

The invention further relates to a composition for topical applicationto skin, comprising S-equol and a vehicle.

The invention further relates to a method of making a compositioncomprising S-equol, comprising the steps of: 1) providing a firstcomposition comprising an isoflavone capable of being converted toS-equol; 2) culturing the first composition with an organism capable ofconverting the isoflavone to S-equol; and 3) incubating the culturedcomposition for a time sufficient to convert a portion of the isoflavoneto S-equol.

The invention additionally relates to a method of making a compositioncomprising S-equol, comprising the steps of: 1) providing a firstcomposition comprising an isoflavone capable of being converted toS-equol; 2) combining the first composition with an enzyme selected fromthe group consisting of: an enzyme that is extracted from a bacteriumcapable of converting the isoflavone to S-equol, an alpha-glucosidase, abeta-glucosidase, beta-galactosidase, gluco-amylase, and pectinase, anda mixture thereof; and 3) incubating the combined composition for a timesufficient to convert a portion of the isoflavone to S-equol.

The invention also relates to a method of making S-equol product,comprising the steps of: 1) providing a composition comprising an equolenantiomer consisting essentially of S-equol, the composition beingproduced in a biological synthesis from the metabolism of an isoflavoneby an organism; 2) extracting S-equol from the composition to form anproduct comprising S-equol, by an extraction selected from: a) a solventextraction, comprising mixing the composition with a low molecularweight alcohol to provide an alcohol to water ratio of at least 40:60and no more than 95:5, and b) an aqueous acid extraction, comprisingmixing the composition at a pH of between about 4.0 and about 5.5; 3)concentrating the extract to a solids content of about 15% to about 55%;4) diluting the concentrate to a solids content of about 6% to about15%; and 5) separating a solid precipitate from the diluted solution;thereby forming the S-equol product.

The invention also relates to a method of delivering S-equol to a mammalto prevent or treat a disease or associated condition, comprisingadministering to the mammal a composition comprising S-equol or aconjugated analog thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of R-equol and S-equol enantiomers.

FIG. 2 shows a chemical reaction scheme wherein formononetin anddaidzein are converted to equol.

FIG. 3 shows the rate of hydrolysis of isoflavone glycosides from soygerm by incubation with enzymes present in Helix pomatia digestivejuice.

FIG. 4 shows a mass chromatogram of the elution of the equol enantiomersfrom a sample of urine from an adult consuming soy food, comparedagainst pure enantiomeric standards that had been characterized byoptical dichroism.

FIG. 5 shows a GC-MS analysis of the trimethylsilyl ether derivative ofsynthesized equol.

FIG. 6 shows another mass chromatogram of a chiral separation of S-equoland R-equol from a racemic mixture.

FIG. 7 shows a mass chromatogram of a chiral separation from anincubation product resulting from the bacterial conversion of daidzeinby intestinal bacteria cultured from an ‘equol-producer’.

FIG. 8 shows a mass chromatogram of a chiral separation from anincubation product resulting from the bacterial conversion of daidzeinby intestinal bacteria cultured from an ‘non-equol-producer’.

FIG. 9 shows the separation and elution of the equol enantiomers from achiral-phase column.

FIG. 10 shows the plasma appearance/disappearance curve for (±)equol ina healthy adult female after oral administration.

FIG. 11 shows estrogenic activity of genistein and (±)equol on theuterus of immature rats.

DETAILED DESCRIPTION OF THE INVENTION

Equol is distinct from most isoflavones in having a chiral center due tothe lack of a double bond in the heterocyclic ring. The phytoestrogenisoflavones from soy, daidzein, glycitein and genistein, from clover,formononetin and biochanin A, and from kudzu, peurarin, do not have achiral center. FIG. 1 shows the chemical structures of R-equol andS-equol.

The R-equol and S-equol enantiomers conformationally differ and this ispredicted to influence how equol fits into the binding site in thecavity of the dimerized ER complex. Many different in vitro assaysystems have been employed to compare the estrogenicity of isoflavones.Independent of the assay system used, data for the relative molarbinding affinities of equol, daidzein, and estradiol to uterinecytosolic receptors are 0.4, 0.1, and 1.0, respectively. These datahowever predate the recognition of distinct ER sub-types and thediscovery of ERβ and therefore the relative binding affinities reflectaffinities toward ERα as this is the predominant receptor in the uterusand take no account of the possible structure-activity differences inthe enantiomeric forms of equol

Several phytoestrogens, including equol, are unique among manyestrogen-like substances for their preferential binding to ERβ proteinand this may serve to explain some of the beneficial effects of soyisoflavones in tissue expressing this receptor sub-type, like the bone,brain and vascular endothelium. More recently, the binding affinity ofequol for human ERα and ERβ was compared with several other isoflavones.The binding of equol to both receptors was similar to that of genistein,but equol induced transcription in gene expression more strongly thanany other isoflavone, especially with ERα. Interestingly, daidzein inthese in vitro systems shows poor affinity and transcriptional activity.

Approximately 50% of equol circulates in the free or unbound form, andthis is considerably greater than the proportion of free daidzein(18.7%) or estradiol (4.6%) in plasma. Since it is the unbound fractionthat is available for receptor occupancy this would effectivelycontribute to enhancing the overall potency of equol. Furthermore,R-equol and S-equol both possess uniquentiandrogen properties by theirability to antagonize dihydrotestosterone in vitro and in vivo, thusexpanding the potential therapeutic role of R-equol as a potentialpharmacological agent in androgen related diseases. R-equol, we predictmay also serve as a ligand for ERβ2 a novel estrogen receptor that mayplay a role in regulating expression of estrogen receptors ERα and ERβand in this regard may prove to be a potential pharmacologic agent forthe treatment or prevention of breast cancer and related hormonalconditions involving signaling pathways mediated through thesereceptors. R-equol also has antioxidant activity. So while R-equol isnot physiological produced in the gastrointestinal tract in response toisoflavone ingestion, it is a unique isoflavone hitherto not recognizedas, and that potentially is, an important pharmacological agent.

As shown in the Experiments section, it was determined that theS-enantiomer of equol is exclusively found in the urine and plasma of“equol-producing” adults consuming soy foods. This suggested thatbacterial production of equol is probably enantiomeric-specific in theintestine, and, as shown in experiment (d) of the Experiments section,S-equol is the only equol enantiomer made by human intestinal bacteriacultured in vitro.

Compositions Containing S-Equol

A composition of the present invention comprises S-equol, and typicallyconsists essentially of S-equol. The composition is used in makingcommercial and institutional products. The composition, or a productmade therefrom, can be consumed orally or applied topically.

The product typically comprises a marketed or institutional foodproduct, a pharmaceutical, an OTC medicament, an ointment, liquid, creamor other material suitable for topical application. A food compositioncan comprise at least 1 mg, and up to 200 mg, S-equol per serving. Anorally-administered medicament can comprise at least 1 mg, and up to 200mg, S-equol per dose.

A product for topical application can comprise at least 0.1%, and up to10%, by weight S-equol. A topical composition of the present inventioncan include other cosmetic and pharmaceutical actives and excipients.Such suitable cosmetic and pharmaceutical agents include, but are notlimited to, antifungals, vitamins, anti-inflammatory agents,antimicrobials, analgesics, nitric oxide synthase inhibitors, insectrepellents, self-tanning agents, surfactants, moisturizers, stabilizers,preservatives, antiseptics, thickeners, lubricants, humectants,chelating agents, skin penetration enhancers, emollients, fragrances andcolorants.

The S-equol can also be an equol conjugate, conjugated at the C-4′ orthe C-7 position with a conjugate selected from the group consisting ofglucuronide, sulfate, acetate, propionate, glucoside, acetyl-glucoside,malonyl-glucoside, and mixtures thereof.

A composition or preparation administered to subjects for the treatingand/or prevention of, or for reducing the predisposition to, diseasesand conditions related thereto can also comprises one or morepharmaceutically acceptable adjuvants, carriers and/or excipients.Pharmaceutically acceptable adjuvants, carriers and/or excipients arewell known in the art, for example as described in the Handbook ofPharmaceutical Excipients, second edition, American PharmaceuticalAssociation, 1994 (incorporated herein by reference). S-equol can beadministered in the form of tablets, capsules, powders forreconstitution, syrups, food (such as food bars, biscuits, snack foodsand other standard food forms well known in the art), or in drinkformulations. Drinks can contain flavoring, buffers and the like.

A composition of the invention can include one suitable for oral,rectal, optical, buccal (for example sublingual), parenteral (forexample subcutaneous, intramuscular, intradermal and intravenous) andtransdermal administration. The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand the state of the patient.

The invention also includes articles of commerce comprising acomposition that comprises a non-racemic mixture of equol, and typicallycomprises equol consisting essentially of S-equol. The article ofcommerce can be a food, including a beverage, and a health or personalcare product.

The composition can typically be made by isolating the S-equolenantiomer from a racemic mixture of R-equol and S-equol (also referredto as (±)equol). Typically, the racemic mixture is a synthetic racemicmixture made by a synthetic route, such as the one described herein.Typically, the S-equol composition has an enantiomeric purity of 90%minimum enantiomeric excess (“EE”) of S-equol. Typically, more purifiedcompositions can be prepared having an EE of 96% minimum, and even moretypically 98% minimum, of S-equol.

The composition of the invention can also comprise a non-racemic mixtureof S-equol and R-equol, having an EE for S-equol of more than 0% andless than 90%. A composition that has an EE of 0% is a 50:50 racemicmixture of the two enantiomers. The composition can be made directlyfrom a racemic mixture, by an incomplete separation and removal ofR-equol enantiomer from the racemic mixture. The composition can also bemade by combining a first equol component comprising a mixture of equolenantiomers, including both a non-racemic mixture and a racemic mixtureof equol, with a second component comprising a composition consistingessentially of S-equol. This produces a non-racemic composition that hasan excess of S-equol. Conversely, a non-racemic mixture can be preparedwith an excess of R-equol enantiomer, by combining a first equolcomponent comprising a mixture of equol enantiomers, including both anon-racemic mixture and a racemic mixture of equol, with a secondcomponent comprising a composition consisting essentially of R-equol.Depending upon the specific benefit or indication for the R-equolcomponent and the S-equol component in a composition, a composition canbe prepared comprising S-equol and R-equol at a ratio of S-equol toR-equol from greater than about 50:50 to about 99.5:1, more typicallyabout 51:49 to about 99:1, and from less than about 50:50 to about1:99.5, more typically about 49:51 to about 1:99.

The S-equol composition can be an additive component of a foodcomposition (which includes also beverages). The food composition cancomprise a probiotic food, a prebiotic food, or a dietary food product.Typically the food product will contain S-equol at a level of from atleast 1 mg per serving size to about 100 mg per serving size, but moretypically 5-50 mg S-equol per serving size.

The food composition of the invention can also comprise S-equol as acomponent of a non-racemic mixture of (±)equol as herein described.

Example compositions according to the present invention can comprise oneor more pharmaceutically-acceptable or industrial standard fillers. Thefiller must not be deleterious to a subject treated with thecomposition. The filler can be solid or a liquid, or both. The fillercan be formulated with the active S-equol as a unit-dose, for example atablet, which can typically contain from about 10% to 80% by weight ofS-equol. Compositions can be prepared by any of the well knowntechniques of pharmacy, for example admixing the components, optionallyincluding excipients, diluents (for example water) and auxiliaries asare well known in the pharmaceutical field.

Compositions suitable for oral administration can be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the extract; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchcompositions can be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active S-equol andone or more suitable carriers (which can contain one or more accessoryingredients as noted above). In general the compositions of theinvention are prepared by uniformly and intimately admixing the S-equolwith a liquid or finely divided solid carrier, or both, and then, ifnecessary, shaping the resulting mixture. For example, a tablet can beprepared by comprising or moulding a powder or granules containing theextract, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine, theextracts in the form of a powder or granules optionally mixed with abinder, lubricant, inert diluents, and/or surface active/dispersingagent(s). Moulded tablets can be made by moulding, in a suitablemachine, the powdered compound moistened with an inert liquid binder.

Suitable fillers, such as sugars, for example lactose, saccharose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example tricalcium phosphate or calcium hydrogen phosphate, and alsobinders such as starch pastes using, for example, corn, wheat, rice orpotato starch, gelatin, tragacanth, methylceullose and/orpolyvinylpyrrolidone, and, if desired, disintegrators, such as theabove-mentioned starches, also carboxymethyl starch, cross linkedpolyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such assodium alginate. Excipients can be flow conditioners and lubricants, forexample silicic acid, talc, stearic acid or salts thereof, such asmagnesium or calcium stearate, and/or polyethylene glycol. Dragee coresare provided with suitable, optionally enteric, coatings, there beingused, inter alia, concentrated sugar solutions which can comprise gumarabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titaniumdioxide, or coating solutions in suitable organic solvents or solventmixtures, or, for the preparation of enteric coatings, solutions ofsuitable cellulose preparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Dyes or pigments can be added tothe tablets or dragee coatings, for example for identification purposesor to indicate different doses of active ingredients.

Other orally administrable pharmaceutical compositions are dry-filledcapsules made, for example, of gelatin, and soft, sealed capsules madeof gelatin and a plasticiser, such as glycerol or sorbitol. Thedry-filled capsules can comprise the extracts in the form of granules,for example in admixture with fillers, such as lactose, binders, such asstarches, and/or glicants, such as talc or magnesium stearate, and,where appropriate, stabilizers. In soft capsules, the extract ispreferably dissolved or suspended in suitable liquids, such as fattyoils, paraffin oil or liquid polyethylene glycols, to which stabilizerscan also be added.

For use in the fortification of food, the S-equol can be mixed with awide range of food products or food components, including cereal,yoghurt, soymilk, soup, cheese, pasta, spread, candy bar, sports bar,drinks, or dairy products.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the extracts in a flavored-base, usually sucrose andacacia or tragacanth; and pastilles comprising the compound in an inertbase such as gelatin and glycerin or sucrose and acacia.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These can be prepared by admixing theisoflavones with one or more conventional solid carriers, for examplecocoa butter, and then shaping the resulting mixture.

Compositions Containing R-Equol

A composition of the present invention can comprise R-equol, andtypically consists essentially of R-equol. The composition is used inmaking commercial and institutional products. The composition, or aproduct or article of commerce made therefrom, can be consumed orally orapplied topically.

The product can comprise any of the products described herein aboverelated to the S-equol, with R-equol at dose levels and compositionlevels that are the same as those for S-equol.

The R-equol can also be an equol conjugate, conjugated at the C-4′ orthe C-7 position with a conjugate selected from the group consisting ofglucuronide, sulfate, acetate, propionate, glucoside, acetyl-glucoside,malonyl-glucoside, and mixtures thereof.

A composition or preparation comprising R-equol that is administered tosubjects for the treating and/or prevention of, or for reducing thepredispositon to, diseases and conditions related thereto can alsocomprises one or more pharmaceutically acceptable adjuvants, carriersand/or excipients, and in the product forms, as described above relatedto the S-equol.

The composition can typically be made by isolating the R-equolenantiomer from a racemic mixture of R-equol and S-equol, as describedabove related to the S-equol.

Identifying Equol Producers and Non-Equol Producers

Studies in healthy adults using [¹³C]daidzein and [¹³C]genistein tracersshow conclusively that equol is formed from daidzein, and not genistein.Equol is formed following the hydrolysis of the glycoside conjugates ofdaidzein from soy, and the methoxylated isoflavone formononetin, or itsglycosidic conjugates found in clover. In all cases the reactionproceeds through a dihydro-intermediate, as shown in FIG. 2. Onceformed, equol appears to be metabolically inert, undergoing no furtherbiotransformation, save phase II metabolism or a minor degree ofadditional hydroxylation in the liver. As with daidzein and genistein,the predominant phase II reactions are glucuronidation and, to a minorextent, sulfation. Following the original discovery that equol'spresence in urine was a function of soy food ingestion, it was observedthat approximately 50-70% of the adult population did not excrete equolin urine even when challenged daily with soy foods, for reasons that areunclear. Furthermore, even when the pure isoflavone compounds areadministered, thereby removing any influence of the food matrix, it hasbeen shown that many people do not convert daidzein to equol. Thisphenomenon has led to the terminology of a person being an‘equol-producer’ or ‘non-equol producer’ (or ‘poor equol-producer’) todescribe these two distinct populations.

Cut-off values have been empirically derived permitting assignment ofindividuals to each of these categories. People who have plasma equolconcentrations of less than 10 ng/mL (40 nmol/L) can be classified as‘non-equol producers’ and where levels are above 10 ng/mL (40 nmol/L)this defines ‘equol producers’. This distinction can also be derivedfrom the levels in urine, an equol producer being someone excretinggreater than 1000 nmol/L. Although the excretion of equol is highlyvariable among individuals there is a large demarcation between thosethat can produce equol and those that cannot, consistent with aprecursor-product relationship in enzyme kinetics catalyzing thereaction. There is consequently an inverse relationship between urinarydaidzein and equol levels, and thus far no significant genderdifferences have been defined.

The status of a subject as an equol producer or a non-equol producer isimportant in the recruitment of subjects for clinical researchevaluating the administering of isoflavones, and particularly daidzein,genistein, formononetin and biochanin A. For example, a number of boneand soy feeding studies have been performed with variable outcomes.Short-term studies, of 12 weeks or less, where surrogate markers ofbone-turnover such as urinary pyridinoline and deoxypyrodinolinecross-links, plasma/serum osteocalcin, alkaline phosphatase, and IGF-1have indicated reduced bone turnover when soy foods containingisoflavones were included in the diet. Several clinical studies ofnine-month duration or less have been reported to show a bone-sparingeffect. All measured changes in bone mineral density (BMD) at varioussites and the results were conflicting, with 2 of the four showing noeffect. In all but one study, there has not been an attempt to defineequol status. In that one study, being an equol-producer was found to beassociated with a significantly increased BMD when soy foods wereconsumed over a 2-year period. Thus, based on our data, equol is abone-trophic agent and therefore identifying a subject as an‘equol-producer’ has therapeutic implications, while delivering equolwill be beneficial in preventing bone loss and increasing boneformation.

The invention includes a method of conducting research wherein anisoflavone is administered to a human subject and at least onephysiological datum is measured, comprising the steps of: 1)administering to at least one human subject of a selected group ofsubjects, a dose of an isoflavone that is a precursor to equol, 2)detecting the level of equol in the urine or blood of the subject, and3) identifying the subject as either an equol producer or a non-equolproducer. The physiological datum is typically one that can be affectedby the estrogenic activity of the isoflavone. Having identified asubject as either an equol producer or a non-equol producer, datacollected from the research study can be analyzed distinctly, wherebythe data of one or more subjects identified as equol producers can becollected, separated, analyzed or reported separately from the data ofone or more subjects identified as non-equol producers. Subjectsidentified as either an equol producer or a non-equol producer can beexcluded from (or included in) the group of research subjects.

Chemical Synthesis of Equol:

In this process standard chemical treatments are used to hydrogenate thedouble-bond of the heterocyclic ring and to remove the carbonyl atposition C-3. Typical starting materials are isoflavones such asdaidzein, genistein, glycitein, peurarin, formononetin and biochanin Aand their glucoside conjugates. Any conjugated form would be reduced toits aglycon by hydrolysis as defined above. Suitable solvents for thereaction include organic acids such as glacial acetic acid, loweralcohols such as isopropanol, and mixtures thereof. Reduction catalyststypically employed include Palladium, such as 10% Pd on charcoal.Reactions can run at temperatures from ambient to 60° C., with pressuresranging from slightly above ambient, up to 200 psig (14 atm. gauge), andwith reaction times of up to 30 hours or more.

After reaction completion, the catalyst is removed and any filtrateevaporated. The crude residue is purified, typically by chromatographyemploying a silica gel column, with an eluent comprising C2-C4 alcohols,C3-C7 alkanes, and mixtures thereof. The purified residue can becrystallized from n-hexane to produce (±)equol as a pure product,typically of at least 99%, with a yield typically of at least 75%. Theequol crystallized product is colorless, not hygroscopic, and stable inair, and does not decompose during the final filtration procedure.

The equol product can be authenticated by GC-MS analysis of thetrimethylsilyl ether, or tert-butyldimethylsilyl ether, or any othervolatile derivative derivative of synthesized product as a single purepeak and a mass spectrum that is consistent with the published electronionization spectrum of the trimethylsilyl (TMS) ether derivative ofauthentic equol.

Method for the Isolation of the Individual R- and S-Enantiomers fromRacemic Equol

The invention also relates to a method of separating a racemic mixtureof equol into its two distinct enantiomers. The method uses a mixture ofracemic equol, typically obtained from a chemical synthesis, as providedabove. A quantity of the racemic equol is introduced into an inlet ofthe HPLC column with a mobile phase comprising a C4-C8 alkyl and a C2-C4alcohol. After a first period of time from passing the racemic mixtureinto the inlet the time period depending upon the type of column, typeof eluent, eluent flow rate, temperature, and mass of the racemicmixture, a first effluent is collected from an outlet of the HPLCcolumn. The first eluent will comprise the first enantiomer, typicallyS-equol. After a second period of time from passing the racemic mixtureinto the inlet, the time period depending upon the type of column, typeof eluent, flow-rate, temperature, and mass of the racemic mixture, asecond effluent is collected from an outlet of the HPLC column. Thesecond eluent will comprise the second enantiomer, typically R-equol.

The separation of equol into S-equol and R-equol can be done on achiral-phase column. A typical example of a chiral-phase column is aChiralcel OD column or OJ column, supplied by Daicel Chemical IndustriesLtd. Columns for separation of marketed quantities of enantiomers can beproduced on industrial systems comprising product and mobile phasepumps, industrial-sized columns, utilities, and control systems. Themobile phase comprises C3-C7 alkanes or a similar polarity solvent,C2-C4 alcohols, and mixtures thereof. The mobile phase typicallycomprises a 95:5 to 5:95, more typically a 50:50 to 90:10, ratio ofhexane to a propanol. A typical example of a mobile phase comprises 70%hexane and 30% ethanol.

The elution of an equol enantiomer from the column can be detected by UVabsorbance at 260-280 nm or by a more specific detection system such asa mass spectrometer and monitoring of ions specific to equol. Theconditions will be optimized to afford complete separation of S-equoland R-equol enantiomers as demonstrated by analytical HPLC.

The chiral-phase column typically comprises a silica substrate to whichis attached a material for selectively separating enantiomers of equol.A typical selection material comprises a cellulosetris(3,5-dimethylphenyl carbamate) and a cellulosetris(4-methylbenzoate).

Biological Production of S-Equol

S-equol can be produced in bulk, and can be produced in situ in avariety of food products, using conventional food technology. A basesolution media, food product or plant extract can be provided thatcomprises daidzein or another related isoflavone from which daidzein canbe derived. The daidzein or other isoflavone can be converted to S-equolby a standard bacterial or enzyme fermentation process, to provide abulk solution, food product or plant extract that comprises S-equol.

The production of S-equol in a food product can be achieved by utilizingthe metabolic activity of bacteria growing on the food that contains asatisfactory starting material, such as daidzin, daidzein, formononetinor peurarin, or a conjugate or mixture thereof. As shown in FIG. 2, theconversion of daidzein to equol involves three major steps: 1)hydrolysis of any glucoside conjugate group, 2) conversion of theisoflavone aglycons to a dihydro-intermediate, and 3) conversion of thedihydro-intermediate to equol. The metabolic pathway and enzymes foreach of the three steps required may not necessarily be present in onebacterium. Anecdotal evidence from human studies suggests that there maybe one or more bacteria that act in conjunction to perform thesereactions, as evidenced from the fact that often dihydrodaidzein can bepresent in significant amounts in plasma and urine yet equol may be lowor barely detectable. Although equol may be produced from daidzein by asingle organism it is believed that better or more efficient conversioncan be achieved when using a mixture of bacterial species, each with itsown metabolic profile. Important conditions for effective conversion toS-equol include the selection of the bacterial organism or mixture oforganisms, the temperature of incubation, and the amount of oxygenavailable to the organisms. These conditions can be optimized bytechniques well known to persons skilled in this art. The organisms usedto effect this change can be inactivated by standard techniques used inthe food industry or, alternately, allowed to remain in an active statein the product.

Bacteria useful in a fermentation process to convert daidzein and/orother structurally related isoflavones, or an intermediate compound, toS-equol, can include a bacterial strain or bacterial strains found tocolonize the intestinal tract of a human, horse, rodent, or other mammalthat is an ‘equol producer’. Since intestinal bacteria in mammals arefound in feces, the equol-producing bacteria can also be found in thefeces of ‘equol producing’ mammals.

Typical bacteria useful in a fermentation process should demonstrate anoptimized conversion rate and extent of conversion that makes thebiological production of equol efficient.

Typically, one or more bacterial strains are required to convert thedaidzein (or other related isoflavone) through intermediate products toS-equol, which generally involves one or more of the three majorreactions: the conversion of isoflavone glycone to aglycon isoflavone;the conversion of aglycon isoflavone to dihydro isoflavone; and theconversion of dihydro isoflavone to the product, equol. For example, amixed culture of organisms isolated from equine feces and a mixedculture of organisms derived from the gastrointestinal tract of a personknown to an ‘equol producer’ can convert, as they do in vivo, theglycone daidzein to the final product S-equol.

Typical bacterial strains that can convert a glycone to an aglycon (suchas daidzein to daidzein) include Enterococcus faecalis, a Lactobacillusplantarum, Listeria welshimeri, a mixed culture of organisms isolatedfrom the intestinal tract of an ‘equol producing’ mammal, Bacteriodesfragilis, Bifidobacterium lactis, Eubactria limosum, Lactobacilluscasei, Lactobacillus acidophilous, Lactobacillus delbrueckii,Lactobacillus paracasei, Listeria monocytogenes, Micrococcus luteus,Proprionobacterium freudenreichii and Sacharomyces boulardii, andmixtures thereof.

Typical bacterial strains that can convert an aglycon to equol (such asdaidzein to S-equol) include Proprionobacteria freundenreichii, a mixedculture containing: Bifidobacterium lactis, Lactobacillus acidophilus,Lactococcus lactis, Enterococcus faecium, Lactobacillus casei andLactobacillus salivarius; and a mixed culture of organisms isolated fromthe intestinal tract of an ‘equol producing’ mammal.

The time required for bacterial conversion of the glucosides toaglycons, or the aglycons to the equol product, will depend uponbacteria-related factors, particularly concentration, the availabilityof oxygen, and the temperature and pH of the incubating system. In mostinstances it is possible to achieve substantially complete conversionwithin 24 hours.

The pH range for bacterial conversion of the isoflavone glucosides toaglycon isoflavones is from about 3 to about 9. The optimum pH dependsprimarily upon the type of bacteria used, and should be selectedaccordingly.

The time required for enzymatic conversion of the glucosides toaglycons, and aglycons to the equol product, depends upon enzyme-relatedfactors, particularly concentration, and the temperature and pH of thesystem. In most instances it is possible to achieve substantiallycomplete conversion within 24 hours, more preferably within about 2hours, and most preferably within about 1 hour.

In an alternative approach to producing equol biologically, S-equol canbe produced in situ in a food product or other suitable substrate by anenzymatic conversion of daidzein or other structurally relatedisoflavone to S-equol. Suitable enzymes can be separated andconcentrated from bacteria that are effective at converting daidzein orstructurally related isoflavones to equol, using standard techniques forseparating and purifying such enzymes. These are well know and used bypractitioners of the art and science of enzymology and biochemistry. Theequol production can be achieved with efficient conversion withoutrequiring growth of bacteria in the food itself.

Enzymes useful in a process to convert daidzein and/or other relatedisoflavones, or an intermediate compound, to equol, can include anenzyme isolated from a bacteria, or a mixture of bacteria, that havebeen shown to convert a suitable isoflavone to equol. An examples ofsuch bacteria or mixtures of bacteria can include, but is not limitedto, bacteria found to colonize the intestinal tract of a human, horse orother mammal that is an ‘equol producer’. Typical enzymes useful in aprocess to convert daidzein or an intermediate compound to equol shoulddemonstrate an optimized conversion rate and extent of conversion thatmakes the biological production of equol efficient.

Enzymes that can be used can be isolated from one or more, or from amixture of, the bacteria described herein for converting daidzein or astructurally related isoflavone, or an intermediate compound, to equol.

In a typical method, bacteria are cultured in a nutritive tryptone brothanaerobically at about 37° C. for about 15 hours to about 72 hours, moretypically from about 24 hours to 36 hours. The bacteria are thenseparated from the culture broth by conventional techniques, mostcommonly by centrifugation at a gravitational force from about 1500 g,up to and in excess of 25,000 g. The cells are washed in saline solution(from about 0.1%, up to about 5%, and preferably at about 0.9%) bysuspending them in the saline, and re-centrifuging the suspension. Thewashed, separated cells are used to prepare an extract of active enzymesusing techniques well know to those practiced in the art of enzymologyand biochemistry. The crude enzyme mixture can be used as-is as anenzyme extract, or can be further purified by conventional enzymepreparation techniques.

A prepared enzyme extract is added to a food containing a suitableisoflavone, such as daidzein, daidzin, or formononetin. Other isolatedenzymes, some of which are commercially available, can be added to speedup the conversion of starting material to intermediates in the enzymemediated reaction pathway. The product is typically incubated at atemperature from about 25° C. to about 45° C., preferably from about 30°C. to about 40° C., while maintaining mild anaerobic conditions in thesamples being grown. The rate of conversion of the daidzein typecompounds to equol is dependent on the amount of active enzyme added tothe food base. Best results are obtained when conversion proceedsrapidly (substantially complete in about 2 hours), but longer times forconversion are necessary at low enzyme activities. The amount of equolproduced in the food may be controlled either by limiting the amount ofdaidzein containing compounds in the food or by inactivating the enzymesat an appropriate time after incubation commences, for example byheating the resulting food product to about 95° to 100° C.

The first step in the enzymatic preparation of S-equol is the conversionof the glucoside to the aglycon. As an alternative to using enzymesisolated in the manner described above to effect this conversion, it ispossible to use commercially available enzymes. The enzymatic conversionof glucosides to aglycons can be performed by bringing a suitable enzymeinto contact with the isoflavone glucosides at a suitable pH andtemperature. The conversion of isoflavone glucosides to aglyconisoflavones has been found to be dependent on a variety of factorsincluding the type of enzymes used, activities of the enzymes, and thepH and temperature of the incubated solution during the conversion. Theenzymes required to effect the conversion are enzymes capable ofcleaving the glucosidic linkage between the isoflavone moiety and theglucose molecule of the isoflavone glucosides. In a preferredembodiment, the enzymes are saccharidase or gluco-amylase enzymescapable of cleaving 1,4-glucoside bonds.

Such enzymes are commercially available alpha- and beta-glucosidaseenzymes, beta-galactosidase enzymes, gluco-amylase enzymes, andpectinase enzymes. Typical examples of these enzymes includeBiopectinase 200AL (which is preferably utilized at a pH range of fromabout 2.5 to about 6.5), available from Deltagen, Redwood City Calif.,Biolactase 30,000 (optimum pH range from about 3 to about 6) NeutralLactase (optimum pH range from about 6 to about 8), both of which areavailable from Quest International, 1833 57th Street, Post Office Box3917, Sarasota, Fla. 34243. Other particularly preferred supplementalenzymes include Lactase NL (optimum pH from about 6 to about 8) andEnzeco Fungal Lactase Concentrate (optimum pH from about 4.5 to about6.5) available from Enzyme Development Corporation, 2 Penn Plaza, Suite1102, 360 West 31^(st) Street, New York, N.Y. 10001; β-galactocidasefrom E. coli (optimum pH from 6.0 to 8.0), manufactured by WorthingtonBiochemicals and available from ScimaR, 4 Ruskin Close, Templestowe,Victoria. 3106, Australia; Lactozyme 3000 L (which preferably isutilized at a pH range from about 6 to about 8), and Alpha-Gal 600 L(which preferably is utilized at a pH range of from about 4 to about6.5), available from Novo Nordisk Bioindustrials, Inc., 33 Turner Road,Danbury, Conn. 06813; Maxilact L2000 (which is preferably utilized at apH range of from about 4 to about 6), available from DSM FoodSpecialties PO Box 1, 2600 MA, Delft, The Netherlands

The pH range for conversion of the isoflavone glucosides to aglyconisoflavones is from about 3 to about 9. The pH that is utilized dependsprimarily upon the type of enzyme used, and should be selectedaccordingly. The enzymes are active within an optimum pH range specifiedby the manufacturer of the enzyme, as shown above for several specificenzymes. Typically the enzymes are active either in a neutral pH rangefrom about 6 to about 8, or in an acidic pH range from about 3 to about6.

The temperature range of an isoflavone-rich material for the conversionof glucosides to aglycons is from about 5° C. to about 75° C. Thetemperature significantly affects the activity of the enzymes, andtherefore, the rate of conversion. The enzymes may be active above 70°C., for example Alpha-Gal 600 L is active at 75° C. However, it ispreferred to conduct the conversion at lower temperatures to avoidenzyme deactivation. In a preferred embodiment, the conversion isconducted at a temperature between about 35° C. to about 45° C.

The time required for conversion of the glucosides to aglycons dependsupon enzyme-related factors, particularly concentration, and thetemperature and pH of the system. In most instances it is possible toachieve substantially complete conversion within 24 hours, however, itis preferred that the enzyme be added to dramatically increase the rateof the reaction. The selected enzyme, enzyme concentration, pH andtemperature preferably cause substantially complete conversion withinabout 2 hours, and most preferably within about 1 hour.

Use of Helix Pomatia as a β-Glucosidase

The invention also relates to a novel method of enyzmaticallyhydrolyzing a glucoside, and in particular an isoflavone glucoside,comprising contacting the glucoside with an enzyme-containing extractfrom Helix pomatia for a time, and under conditions, sufficient toconvert the glucoside to the corresponding aglycon. Theenzyme-containing extract is typically the digestive juice of HelixPomatia.

In the course of synthesizing equol from its isoflavone glucosidestarting material, it was discovered that the digestive juice of Helixpomatia effectively serves as a β-glucosidase for converting anisoflavone glucoside to the aglycon isoflavone. Helix pomatia digestivejuice is commercially marketed as a β-glucuronidase and sulfatasepreparation and has for thirty years been the enzyme preparation ofchoice for hydrolysis of steroid and isoflavone conjugates. Its use as aβ-glucosidase was unknown and unexpected. The β-glucosidase activity issufficiently capable of completely hydrolyzing in vitro isoflavoneconjugated with sugar moieties.

The digestive juice can be used as-is, or in a purified form. Theefficiency of Helix pomatia digestive juice to hydrolyze isoflavoneglycosides was established by incubating in vitro 100 μg each of daidzinand genistin with 0.1 mL of Helix pomatia digestive juice suspended in10 mL of 0.05M sodium acetate buffer, pH 4.5 at 37° C. Before adding theenzyme/buffer mixture, it was passed through a solid-phase C₁₈ Bond Elutcartridge to remove residual amounts of isoflavones that we havepreviously found to naturally occur in this enzyme preparation. Theconcentrations of daidzin and genistin remaining, and daidzein andgenistein formed during incubation were determined by HPLC on aliquotsof the mixture removed at timed intervals over the next 24 hours.

FIG. 3 shows the time course for the hydrolysis of daidzin and genistinby Helix pomatia as measured by HPLC from the proportion of glycosidesto aglycons remaining in the incubation mixture. These in vitro studiesshow that under the analytical conditions employed Helix pomatiacompletely hydrolyzed daidzin and genistin within 15 min and this enzymepreparation in addition to having β-glucuronidase and sulfatase activityis also a useful source of β-glucosidases.

Separation of S-Equol from Bulk Solution

S-equol produced in bulk can be separated from the resulting bulksolution of a bacterial or enzymatic production of S-equol, by methodswell known in the art, including crystallization, solvent extraction,distillation, and precipitation/filtration. The resulting bulk solutioncan contain unreacted daidzein or other related isoflavone used,by-products, and any reactants. Such methods can include the use of areverse-phase or straight-phase liquid chromatography column and thesecan be combined with chiral-phase chromatography

A typical method of removing S-equol from a bulk solution or solid phaseis by extraction. An extractant solution is added to the solution orsolid phase containing the S-equol. Typically the extractant is a lowmolecular weight alcohol such as methanol, ethanol, isopropyl alcohol,or propyl alcohol, or an aqueous solution having a pH in the range from3.5 to 5.5. Typically, if the aqueous alcohol method is being used,sufficient alcohol is added to bring the alcohol/water ratio to betweena minimum of 40:60 and a maximum of 95:5. More typically, the ratio isat least 60:40, and even more typically a ratio between 65:35 and 90:10.

If an aqueous acid extraction method is used an aqueous acid solution isprepared with the pH adjusted to about 3.5 to about 5.5, and morepreferably within the pH range of about 4.0 to about 5.0. Sufficientwater is added to make a dilute liquid with a sufficiently low viscosityto permit separation of solids from liquids by centrifugation orfiltration.

The liquid, from which insoluble solid matter has been removed, isconcentrated by conventional methods for removing liquids. Methods usedtypically include, but are not limited to, removal of solvent byevaporation, preferably under reduced pressure. The residual liquid isconcentrated to at least about 15% solids, and up to about 55% solids,more typically to between 30% and 50% solids. The concentrate is thendiluted with water to reduce the solids content and increase the waterto alcohol ratio. The amount of water added can be varied over a widerange, though a final solids content between 6% and 15%, and moretypically about 13%, is preferred. The pH of the mixture is adjustedbetween about pH 3.0 and about pH 6.5, with a preferred value betweenabout pH 4.0 and about pH 5.0. Typically the temperature is betweenabout 2° C. to about 10° C., and more typically about 5° C. to 7° C.

The solid material is then separated from the liquid by standardseparation techniques (centrifugation or filtration) and yields anequol-rich solid material.

The equol-rich material can optionally be purified, typically bychromatography employing a silica gel column, with an eluent comprisingC2-C4 alcohols, C3-C7 alkanes, and mixtures thereof. The purifiedresidue can be crystallized from n-hexane to produce S-equol as a pureproduct, typically of at least 99%, with a yield typically of at least75%. The equol crystallized product is colorless, not hygroscopic, andstable in air, and does not decompose during the final filtrationprocedure.

The S-equol product can be authenticated by GC-MS analysis of thetrimethylsilyl ether or tert-butyldimethylsilyl ether derivative, orsome other appropriate volatile derivative of synthesized product as asingle pure peak and a mass spectrum that is consistent with thepublished electron ionization spectrum of the trimethylsilyl (TMS) etherderivative of authentic equol. Confirmation of the product can also beestablished by direct mass spectrometry using electrospray ionizationafter introducing the sample into the instrument via an HPLCchiral-phase column.

Treatment of Disease by Administering S-Equol

This present invention provides a means for an individual subject toovercome the problem of not being able to produce equol in vivo, byproviding delivery of equol enantiomers, and specifically S-equol ormixtures of S-equol and R-equol directly, circumventing the need forintestinal bacteria for its production. The delivery of S-equol can alsosupplement the in vivo production of S-equol in ‘equol-producers’, aswell as in ‘non-equol producers’.

This invention provides a method for delivering S-equol in sufficientamounts to have health benefits. The active S-equol material can bedelivered by direct ingestion or administration of the pure S-equolcompound or any conjugated analog of S-equol. Typically, the amount ofcomposition comprising S-equol is administered in an amount sufficientto produce a transient level of S-equol in the blood plasma of themammal of at least 5 nanograms per milliliter (ng/mL), more typically atleast 10 ng/mL or greater. or transient levels of S-equol in urine ofgreater than 1000 nmol/L. The S-equol can also be an S-equol conjugate,conjugated at the C-4′ or the C-7 position with a conjugate selectedfrom the group consisting of glucuronide, sulfate, acetate, propionate,glucoside, acetyl-glucoside, malonyl-glucoside, and mixtures thereof.Typically, the composition is administered orally in a dose amount of atleast about 1 mg, more typically of at least 5 mg, and of up to 100 mg,more typically, up to 50 mg.

The ability to deliver the S-equol in sufficient amounts is believed toprovide several advantages over delivery of a racemic mixture of equol.First, the potency of S-equol is expected to be at least twice that ofthe racemic mixture. Second, the human body only produces the S-equol,and therefore, a composition comprising only S-equol represents a“natural” product with an ingredient, S-equol, with which the body isfamiliar. And third, since it is believed that the R-equol enantiomer isnot produced by the human body, a treatment composition comprising only,or substantially only, the S-enantiomer does not introduce a materialwith which the body is unfamiliar.

Compositions of the present invention can be used to treat a variety ofhormone-dependent diseases and conditions associated therewith.

The invention includes the use of S-equol to treat and prevent diseasesand conditions including brain disorders, dementia of the Alzheimertype, as well as other reduced or impaired cognitive functionsassociated with advancing age and with short- and long-term memory loss.The estrogenic activity of S-equol acts in the brain by enhancingneurotransmission and restoring synaptic density. It is believed thatS-equol is active in the brain at the same site as estrogen, exerting anestrogenic response.

The invention includes the use of S-equol to treat and preventosteopenia and osteoporosis.

In a two-year randomized study, postmenopausal women consumed each daytwo glasses of soymilk, either with or without isoflavones. The datafound that lumbar spine BMD and BMC decreased 4.0% and 4.3%,respectively (p<0.01) over the 2 year period in the group consumingsoymilk with negligible amounts of isoflavones. These levels are closeto the 5-7% loss in bone mass that would be normally expected in thefirst two years of natural menopause. By contrast, those women consumingsoymilk that contained 50 mg isoflavones showed an increase of 1.1% and2% in lumbar spine BMD and BMC respectively (relative to baselinevalues). This study showed that soy protein with isoflavones, as opposedto lacking in isoflavones, maintained stable bone mass over a 2-yearperiod. The data suggests that bone loss as measured by changes inlumbar spine BMD was prevented by the presence of isoflavones.

It should be mentioned that this difference was not observed after onlyone year. Given the slow rate of bone turnover the variability in datafrom previous bone studies is likely to be a consequence of the shortduration of dietary intervention with soy foods.

The most striking observation was that women who were ‘equol-producers’,defined by a plasma equol concentration of greater than 10 ng/mL (45% ofthe cohort), showed mean increases of 2.4% and 2.8% respectively forbone mineral density (BMD, p=0.02) and bone mineral content (BMC,p=0.009) in the lumbar spine, compared with increases of only 0.6% and0.3%, respectively in women in the ‘non-equol producing’ group. Womenadministered a control substance showed mean decreases of 4.0 and 4.3%,respectively (p≦0.01 compared to baseline). This data demonstrates thatthe ability to metabolize isoflavones to produce equol, and the presenceof equol in the body, have a direct relation to increased BMD and BMC.These data suggest that equol is an important bone-trophic agent. Thecomposition comprising S-equol is administered in an amount sufficientto reduce the surrogate markers of bone turnover, or prevent bone lossas measured by bone mineral density. The composition comprising S-equolcan also be administered in an amount sufficient to increase boneformation, or to prevent osteoporosis and reduce bone fracture.

The invention includes the use of S-equol to treat and prevent lipiddisorders such as high cholesterol (hypercholesterolemia), lipidemia,lipemia and dyslipidemia (disturbances in lipids). The study describedabove also included the study of the cholesterol concentrations in thetest subjects. The results showed that plasma total cholesterolconcentrations decreased 7.2% (p=0.04) in equol producers compared withbaseline levels and 3.0% (p=NS) in non-equol producers. The failure ofsoy protein to have significant cholesterol-lowering effects in adultswith normal blood cholesterol levels, is, with few exceptions, probablybecause of heterogeneity in the study populations with regard to themetabolism of soy isoflavones and the failure to recognize the relevanceof equol formation. These data suggest that equol influences lipids in afavorable manner. The composition comprising S-equol is administered inan amount sufficient to reduce the level of lipids in the blood stream.

The invention also includes the use of S-equol to treat and preventacute and chronic ovarian estrogen deficiency states including,vasomotor disturbances and night sweats, commonly referred to as ‘hotflushes’ or ‘hot flashes’. This also includes hot flushes accompanyingantiestrogen therapy used in the treatment of breast cancer.

The invention also includes the use of S-equol to treat and preventcardiovascular disease and liver disease.

The invention further includes the use of S-equol to improve diminishedblood vessel quality, by increasing reactivity or flexibility inresponse to acute changes in blood pressure, improving blood flow, andreducing blood pressure.

The invention includes the use of S-equol to reduce lipid peroxidationand act as an antioxidant in scavenging free-radicals in the body.

The invention also includes the use of S-equol to reduce inflammation asevidenced by effects on reducing markers of inflammation such asC-reactive protein.

The invention also includes the use of S-equol to treat and preventcancer, including benign breast cancer, breast cancer, benign prostatecancer, prostate cancer, skin cancer, and colon cancer.

The invention also includes the use of S-equol to treat and preventadenomatous polyps and familial polyposis, both of which are high-riskconditions predisposing to colon cancer. Given the important role ofestrogen in reducing colon cancer risk in women, it is reasonable toexpect enantiomers of equol to have similar preventive or therapeuticactions, especially as the colon is the major site of equol productionfrom its precursors.

Compositions of the present invention can be used to treat a variety ofnon-hormone-dependent diseases and conditions associated therewith,including inflammatory conditions of the gastrointestinal tract, theprostate, the breast, the skin and bone.

The presence of a chiral center in the equol molecule may have relevanceto its biological potency. The efficacy of the enantiomers will begreater than the racemate, especially toward ERβ.

In the method of this invention, calcium, or vitamin D can beco-administered (that is before, at the same time or after the S-equol),for example as a separate tablet, or as part of a suitable dosage form.

Equol possesses other properties of relevance to cellular function.Being a polyphenol it shares with flavonoids the ability to be ahydrogen/electron donor and therefore may scavenge free radicals. Equolhas the greatest antioxidant activity of all the isoflavones tested whenmeasured in vitro in the FRAP, TEAC and Cu(II)-induced orferric(III)-induced liposomal peroxidation assays. Although isoflavonesare considered weak antioxidants when tested in vitro, their in vivoeffect may be significant enough to account for the reduced ex vivolipid peroxidation that has been observed in all but one clinical studywhen adults consume soy protein diets. Given the superior antioxidantactivity of equol over other isoflavones, a case can be made for beingan ‘equol-producer’ or in those people unable to make equol fordelivering directly equol either as a pharmacologic agent, a supplementor in a food product. In all cases enhanced circulating equol levels canprovide greater inhibition of lipid peroxidation and therefore greaterreduction in risk for cardiovascular disease.

It is believed that non-equol producers are generally at a higher riskthan equol producers for developing certain diseases, typicallyhormone-dependent diseases or conditions, including breast cancer. Forthose humans who are poor- or non-equol-producers, comparable benefitsmay be attained by oral, topical, nasal, subcutaneous, or intravenousadministration of equol enantiomers or mixtures thereof.

Supplementing the diet of the equol producers with equol, andparticularly the S-equol, can provide benefits when the ordinary levelof S-equol produced by the equol producer is inadequate because of 1)insufficient consumption of isoflavones to produce equol, 2) antibioticuse that wipes out the activity of intestinal bacteria to make equolfrom precursor isoflavones, or 3) other health factors that impact thelevel of equol production. In addition, a supplemental level of equol,and particularly S-equol, is believed to provide enhanced effect on thehealth and well-being of the person.

Treatment of Disease by Administering R-Equol

This present invention also provides a means for an individual subjectto overcome the problem of not being able to produce equol in vivo, byproviding delivery of equol enantiomers, and specifically R-equol ormixtures of R-equol and S-equol directly, circumventing the need forintestinal bacteria for its production.

This invention provides a method for delivering R-equol in sufficientamounts to have health benefits. The R-equol material can be deliveredby direct ingestion or administration of the pure R-equol compound orany conjugated analog of R-equol. Typically, the amount of compositioncomprising R-equol is administered in an amount sufficient to produce atransient level of R-equol in the blood plasma of the mammal of at least5 nanograms per milliliter (ng/mL), more typically at least 10 ng/mL orgreater, or a transient level of R-equol in urine of greater than 1000mmol/L. The R-equol can also be an equol conjugate, conjugated at theC-4′ or the C-7 position with a conjugate selected from the groupconsisting of glucuronide, sulfate, acetate, propionate, glucoside,acetyl-glucoside, malonyl-glucoside, and mixtures thereof. Typically,the composition is administered orally in a dose amount of at leastabout 1 mg, more typically of at least 5 mg, and of up to 100 mg, moretypically up to 50 mg.

The ability to deliver R-equol in sufficient amounts is believed in somecircumstances, such as breast cancer prevention, or for antagonizingligand binding to specific ER such as ERβ1 or ERβ2, to provide someadvantage over delivery of a racemic mixture of equol.

Compositions of the present invention comprising R-equol can be used totreat a variety of hormone-dependent diseases and conditions associatedtherewith.

The invention includes the use of R-equol to treat and prevent diseasesand conditions including brain disorders, dementia of the Alzheimertype, as well as other reduced or impaired cognitive functionsassociated with advancing age and with short- and long-term memory loss.It is believed that R-equol is active in the brain at the same site asestrogen, exerting an estrogenic response mediated through specificestrogen receptors that are rich in certain regions of the brain, whilealso having antioxidant effects in protecting neurons against oxidativestress.

The invention includes the use of R-equol to treat and preventosteopenia and osteoporosis since antioxidants have protective effectsagainst osteoclast activity.

The composition comprising R-equol is administered in an amountsufficient to reduce the surrogate markers of bone turnover, or preventbone loss as measured by bone mineral density. The compositioncomprising R-equol can also be administered in an amount sufficient toincrease bone formation, or to prevent osteoporosis and reduce bonefracture.

The invention includes the use of R-equol to treat and prevent lipiddisorders such as high cholesterol (hypercholesterolemia), lipidemia andlipemia or dyslipidemia (disturbances in lipids). The failure of soyprotein to have significant cholesterol-lowering effects in adults withnormal blood cholesterol levels is, with few exceptions, probablybecause of heterogeneity in the study populations with regard to themetabolism of soy isoflavones and the failure to recognize the relevanceof equol formation. These data suggest that equol influences lipids in afavorable manner. The composition comprising R-equol is administered inan amount sufficient to reduce the level of lipids in the blood streamand to reduce lipid peroxidation.

The invention also includes the use of R-equol to treat and preventacute and chronic ovarian estrogen deficiency states including,vasomotor disturbances and night sweats, commonly referred to as ‘hotflushes’ or ‘hot flashes’. This also includes hot flushes accompanyingantiestrogen therapy used in the treatment of breast cancer.

The invention also includes the use of R-equol to treat and preventcardiovascular disease and liver disease.

The invention further includes the use of R-equol to improve diminishedblood vessel quality, by increasing reactivity or flexibility inresponse to acute changes in blood pressure, improving blood flow, andreducing blood pressure.

The invention includes the use of R-equol to act as an antioxidant inscavenging free-radicals in the body.

The invention includes the use of R-equol to reduce inflammation asevidenced by effects on reducing markers of inflammation such asC-reactive protein and cytokines.

The invention also includes the use of R-equol to treat and preventcancer, including benign breast cancer, breast cancer, benign prostatecancer, prostate cancer, skin cancer, and colon cancer.

The invention also includes the use of R-equol to treat and preventadenomatous polyps and familial polyposis, both of which are high-riskconditions predisposing to colon cancer. Given the important role ofestrogen in reducing colon cancer risk in women, it is reasonable toexpect enantiomers of equol to have similar preventive or therapeuticactions, especially as the colon is the major site of equol productionfrom its precursors.

Experiments

(a) Determination of Equol Enantiomer in ‘Equol-Producing’ Adults

The urine samples from adults consuming soy foods previously identifiedas being ‘equol-producers’ were analyzed. Equol was isolated from urine(25 mL) by passage of the sample through a solid-phase Bond Elut C18cartridge. After washing the cartridge with water, the isoflavones wererecovered by elution with methanol (5 mL) and the methanolic phase wastaken to dryness under a stream of nitrogen. The sample was subjected toenzymatic hydrolysis with Helix pomatia and then re-extracted on a BondElut C18 cartridge. The methanolic extract was taken to dryness undernitrogen gas and redissolved in HPLC mobile phase (100 μL). Equolenantiomers were identified by HPLC using a Chiralcel OJ chiral phasecolumn using the method shown in Example 2. The detection of equol wasachieved by selected ion monitoring electrospray ionization massspectrometry (ESI-MS). Mass chromatograms of a pure standard of S-equol,and of urine from an adult consuming soy food are shown in FIG. 4.

The retention index and mass chromatogram establish that it isexclusively the S-enantiomer of equol that is excreted in human urine asno detectable R-enantiomer of equol could be found. Analysis of theplasma from the same ‘equol-producer’ also revealed only theS-enantiomer of equol.

(b) Chemical Synthesis of Racemic Equol

Daidzein (200 mg, 0.8 mmol) is dissolved in a mixture of glacial aceticacid (20 mL) and isopropanol (20 mL), and is reduced with 10% Pd oncharcoal (150 mg) at 55 p.s.i.g. (3.7 atm gauge). At the end of thereaction (2 hours, TLC:isopropanol/n-hexane 1/4) the catalyst isfiltered off, and the filtrate is evaporated. The crude residue ispurified by chromatography on a silica gel column using as eluent amixture of isopropanol and n-hexane (1:4 v/v), to give (±)equol as apure product (160 mg, yield: 82%) crystallized from n-hexane. Theproduct, colorless crystals, is not hygroscopic, is stable in air, anddoes not decompose during the final filtration procedure. The product ofthis chemical synthesis was in all respects identical with an authenticsample of (±)equol (racemic equol). FIG. 5 shows the GC-MS analysis ofthe trimethylsilyl ether derivative of synthesized product as a singlepure peak and a mass spectrum that is consistent with the publishedelectron ionization spectrum of the trimethylsilyl (TMS) etherderivative of authentic equol. The molecular ion as expected is at m/z470 and the base peak at m/z 234. The purified equol product had apurity of greater than 99%, as confirmed by HPLC and mass spectrometry.

(c) Elution order of S- and R-Enantiomer by Optical Dichroism

A racemic mixture of S-equol and R-equol were separated by chiralchromatography on a Chiralcel OJ Column using a flow-rate of 1.0 mL/minand with a gradient elution consisting of an initial mobile phase of 10%ethanol in hexane and increasing to 90% ethanol in hexane over a timeperiod of 15 minutes according to the program shown in Table A:

TABLE A Time (min.) % hexane % ethanol 0 90 10 1.0 90 10 15.0 10 90 16.090 10 17.0 90 10

FIG. 6 shows the mass chromatogram of the ions recording (m/z 241) for aracemic mixture of S- and R-equol.

The first eluting material, designated as Enantiomer-1, and the secondeluting material, designated as Enantiomer-2, were collected separately.Each enantiomer was weighed and the weighed samples dissolved in 1 mL ofspectroscopic grade ethanol. Measurement of the optical rotation of eachenantiomer was carried out at 20° C. using the light of wavelength inthe line D of sodium.

Enantiomer-1 material (1.6 mg exact weight) had first and secondmeasurements of −0.023 and −0.022, resulting in an optical rotation of−14 [−0.0225×1000/1.6], which corresponds with the S-enantiomer ofequol. Enantiomer-2 material (1.7 mg exact weight) had first and secondmeasurements of +0.023 and +0.023, resulting in an optical rotation of+13.5 [+0.023×1000/1.7], which corresponds with the R-enantiomer ofequol.

(d) Production of S-Equol by Human Intestinal Bacteria

Freshly voided feces (1 g) from an equol-producer and a non-equolproducer were separately incubated with 9 mL of sterile distilled water,trypticase soy broth and brain-heart infusion broth with the addition ofdaidzein (10 mg/L). The broths were incubated anerobically at 37° C. for24 hr. The incubation mixtures were then centrifuged and the isoflavonesisolated by passage through a Bond Elut C18 solid-phase cartridge(Varian Inc, Harbor City, Calif.), and eluted with methanol. Themethanolic extract was then taken to dryness over nitrogen gas andre-dissolved in 100 μL mobile phase for analysis by high pressure liquidchromatography coupled with electrospray ionization mass spectrometry(ESI-MS).

The sample extracts (20 μL) were injected on column using the chiralphase column and elution properties described above in experiment (c).Detection of the two enantiomers was accomplished by selected ionrecording in negative ion mode of the ion at m/z 241 specific for bothequol enantiomers. The mass chromatograms of the incubation extractswere compared with a pure standard of the racemic equol that containedapproximately equal proportions of S-equol and R-equol. Identificationwas based on the retention differences between the two enantiomers,where the S-equol enantiomer eluted before the R-equol enantiomer.

FIG. 7 shows the mass chromatogram of the ions recording (m/z 241) forthe incubation product resulting from the bacterial conversion ofdaidzein to equol by intestinal bacteria cultured from the‘equol-producer’. FIG. 7 shows a significant peak that corresponds tothe S-equol enantiomer. By contrast, FIG. 8 shows the mass chromatogramof the ions recording (m/z 241) for the incubation product resultingfrom the bacterial conversion of daidzein to equol by intestinalbacteria cultured from the ‘non-equol-producer’, indicating that S-equolwas detected in trivial or trace levels from its minor peak at theretention time corresponding to S-equol.

The product formed from intestinal bacterial conversion of daidzein toequol was a single peak (Enantiomer-1) corresponding to exclusively theS-equol enantiomer based upon ESI-MS analysis.

These studies confirm that human intestinal bacteria exclusively producethe S-equol enantiomer and this is consistent with the appearance ofS-equol in human plasma and urine.

e) Determination of Receptor Binding Capacity of S- and R-Enantiomers

In vitro binding studies were performed to examine the relativeaffinities of S- and R-enantiomeric equol with the estrogen receptorsERα and ERβ.

Synthesis of Hormone Receptor Proteins: Full length rat ERα expressionvector (pcDNA-ERα; RH Price UCSF) and ERβ expression vector (pcDNA-ERβ;TA Brown, Pfizer, Groton, Conn.) were used to synthesize hormonereceptors in vitro using the TnT-coupled reticulocyte lysate system(Promega, Madison, Wis.) with T7-RNA polymerase, during a 90 minreaction at 30° C. Translation reaction mixtures were stored at −80° C.until further use.

Saturation isotherms: In order to calculate and establish the bindingaffinity of the S-equol and R-equol enantiomers for ERα and ERβ, 100 μLaliquots of reticulocyte lysate supernatant were incubated at optimaltime and temperature; 90 min at room temperature (ERβ) and 18 hrs at 4°C. (ERα), with increasing (0.01-100 nM) concentrations of [³H]17β-estradiol (E₂). These times were determined empirically andrepresent optimal binding of receptor with estrogen. Nonspecific bindingwas assessed using a 300-fold excess of the ER agonist,diethylstilbestrol, in parallel tubes. Following incubation, bound andunbound [³H]E₂ were separated by passing the incubation reaction througha 1 mL lipophilic Sephadex LH-20 (Sigma-Aldrich Co., Saint Louis, Mo.)column. Columns were constructed by packing a disposable pipette tip (1mL; Labcraft, Curtin Matheson Scientific, Inc, Houston, Tex.) with TEGMD(10 mM Tris-Cl, 1.5 mM EDTA, 10% glycerol, 25 mM molybdate, and 1 mMdithiothreitol, pH 7.4)-saturated Sephadex according to previouslypublished protocols (Handa et al., 1986; O'Keefe and Handa, 1990). Forchromatography, the columns were re-equilibrated with TEGMD (100 μL),and the incubation reactions were added individually to each column andallowed to incubate on the column for an additional 30 min. Followingthis incubation, 600 μL of TEGMD were added to each column, flow-throughwas collected, 4 mL scintillation fluid was added, and samples werecounted (5 min each) in an 2900 TR Packard scintillation counter(Packard Bioscience, Meriden, Conn.).

Competition binding studies were used to assess the estrogenicproperties of equol's S-equol and R-equol enantiomers. Based on theability of S and R to compete with [³H] E₂ for ER binding, theaffinities for in vitro translated ER were shown to be very differentfor the two enantiomers. The S-equol enantiomer showed greatest affinityfor ERβ [Kd (nM)=0.73±0.2], while its affinity for ERα was relativelylow by comparison [K_(d)(nM)=6.41±1.0]. The R-equol enantiomer possesseda much lower affinity for both ERβ [Kd (nM)=15.4±1.3] and ERα [Kd(nM)=27.38±3.8]. For reference 17β-estradiol binds ERα with a Kd(nM)=0.13 and ERβ with a Kd (nM)=0.15 in this system.

The study shows that only the S-equol enantiomer binds ER withsufficient affinity to have potential relevance to circulating equollevels reported in humans. Compared with 17β-estradiol the relativebinding affinities of the S-equol and R-equol enantiomers for ERα were49-fold and 211-fold less, respectively. However, the S-equol enantiomerseems to be largely ERβ-selective with a relatively high affinity forERβ, while the R-equol enantiomer binds with approximately 100-fold lessaffinity. The separate and associated determination that exclusivelyS-equol is found in human plasma and urine is significant in view of thespecificity in binding of the two enantiomers.

EXAMPLES Example 1 Separation of Racemic Equol into Separate Enantiomersby HPLC

A synthetic racemic mixture of S- and R-equol was prepared in accordancewith the chemical synthesis described in experiment (b) in theExperiments section, and was passed through a Chiralcel OJ (0.46 cmdiameter×25 cm long), supplied by Daicel Chemical Industries Ltd. Thecolumn uses cellulose tris(4-methylbenzoate) on a 10 μm silica-gelsubstrate. The mobile phase used was a gradient elution beginning withhexane 90%/ethanol 10% and linearly increasing to a final composition ofhexane 10%/ethanol 90% over a 15 min period according to Table A at aflow-rate of 1 milliliters per minute (mL/min). The elution of equolfrom the column was detected by UV absorbance at 260 nm. FIG. 9 showsthe elution of equol enantiomers using the chiral-phase column. R-equolenantiomer had a residence time of 7.05 min., while the S-equolenantiomer had a residence time of 7.75 min. The identification of theenantiomers was confirmed from their retention indices and comparisonagainst pure enantiomeric standards that had been characterized byoptical dichroism.

Example 2 Absorption and Bioavailability of Equol

A healthy adult human female subject was administered a single bolusoral 25-mg dose of equol, and blood plasma levels of equol aremonitored. Absorption through the intestinal tract proceeded rapidly,attaining a maximal plasma concentration after 4-6 hr, and thereafterdisappearing from the circulation with a terminal elimination half-lifeof 8.8 hr. The pharmacokinetics of ±equol, shown in Table B, are similarto those of other isoflavones, although showing a slower plasmaclearance (Cl/F=6.85 L/h) compared with its precursor, daidzein(Cl/F=17.5 L/h), and showing a relatively high dose adjustedbioavailability (AUC inf/F=145.8 ng/Ml/hr/mg equol). FIG. 10 shows theplasma appearance/disappearance curve for (±)equol expressed aslog/linear plot depicting equol's pharmacokinetics in the healthy adultfemale after oral administration of (±)equol. Table B also showscomparative values previously published for daidzein in healthy women.

TABLE B Adult Female Equol Daidzein t½ (h) 8.76 9.34 Vd/F (L) 86.7 236.4Cl/F (L/h) 6.85 17.5 AUC_(inf) (ng/Ml/hr) 3646 1470

Example 3 Estrogenic Activity of Equol

A racemic mixture of a chemically-synthesized equol was sub-cutaneouslyinjected (both 100 mg and 500 mg doses) into prepubertal 22-day oldSprague-Dawley rats to compare its estrogenic activity on the uterus ofimmature rats. Also tested were genistein (500 mg dose) and DMSO(control). Uterine weights were measured on days 17, 19, and 21. FIG. 11shows that the racemic equol was more than twice as estrogenic thangenistein in this model when allowing for the fact that half of theinjected dose is the inactive R-equol enantiomer.

Example 4 Bacterial Conversion of Glucoside to Aglycon Forms in SoyFoods

The first step in the conversion of daidzein to equol in foods is theconversion of the glucoside form of the isoflavone to the aglycon formin preparation for the enzymic reduction of the aglycon to equol. Alarge number of organisms were tested for their ability to achieve theconversion. Sterile soy beverage containing approximately 3.5% protein,8% carbohydrate and daidzin at approximately 16 mg/L was inoculated witha test organism and incubated at a suitable temperature. Incubationtemperatures in the range of 200 to 40° C. were considered suitable and,for most of the organisms tested, a temperature of 30° or 37° waspreferred. Incubation was carried out under anaerobic conditions in themajority of the bacterial strains. The progress of the conversion ofdaidzin to daidzein was followed by analyzing samples for unreacteddaidzein, taken at intervals from 10 hours up to 72 hour afterincubation commenced. The results are shown in Table C. Of fifty fourspecies/strains of bacteria tested, there were 26 which were unable toconvert daidzin to daidzein. Of the 28 organisms that were able toconvert daidzin to daidzein, four were able to do the conversionrapidly, taking from 10 hours to 24 hour to achieve virtually 100%conversion. There were twelve types that converted at a medium rate,taking from 25 to 72 hours to achieve virtually complete conversion. Theremaining organisms that provided conversion were slow, with less than50% conversion being completed within the 72 hour incubation period. Theorganisms showing rapid conversion included Enterococcus faecalis, aLactobacillus plantarum, Listeria welshimeri, and a mixed culture oforganisms isolated from equine feces Of seven Lactobacillus plantarumstrains tested, one was fast, four classified as medium and two wereslow convertors. Other organisms able to convert efficiently theglucoside to the aglycon included Bacteriodes fragilis, Bifidobacteriumlactis, Eubactria limosum, Lactobacillus casei, lactobacillusacidophilous, Lactobacillus delbruekii, Lactobacillus paracasei,Listeria monocytogenes, Micrococcus luteus, Proprionobacteriumfreudenreichii and Sacharomyced boulardii.

TABLE C Rate of Conversion of Daidzin to Daidzein by VariousMicroorganisms in a Food Base (Incubation at 37° C. under AnaerobicConditions) Time to Time to reach 50% reach >90% conversion conversionBacterial species/strain (hours) (hours) Bacteroides fragilis 64 NDBifidobacterium lactis Bb-12 (ChB) 25 >40 Bifidobacterium lactis STSC380 (lafti) 40 ND Enterococcus faecalis STSC 030 5 8 Eubacterium limosum35 >64 Mixed culture from equine feces 8 15 Lactobacillus acidophilusSTSC 220 (GbA) 25 40 Lactobacillus acidophilus STSC 375 28 64Lactobacillus casei STSC 175 25 40 Lactobacillus casei STSC 330 30 64Lactobacillus casei STSC 355 25 64 Lactobacillus delbruekii STSC 350 40ND Lactobacillus paracasei STSC 385 22 48 Lactobacillus paracasei STSC345 30 48 Lactobacillus paracasei ChC 17 25 Lactobacillus plantarum STSC300 Lp 429 16 24 Lactobacillus plantarum Lp 2904 27 >30 Lactobacillusplantarum Lp 1572 27 >30 Lactobacillus plantarum STSC 325 48 NDLactobacillus plantarum STSC 335 64 ND Lactobacillus plantarum Lp 737625 60 Lactobacillus plantarum Lp 704 27 40 Listeria monocytogenes STSC135 27 40 Listeria welshimeri STSC 260 15 24 Micrococcus luteus STSC 37050 ND Mixed culture containing: Bifidobacterium 18 32 lactis,Lactobacillus acidophilus, Lactococcus lactis, Enterococcus faecium,Lactobacillus casei and Lactobacillus salivarius. Propionibacteriumfreudenreichii 18 22 Saccharomyces boulardii ATCC 74012 64 ND ND -Unable to estimate time >values were approaching the category threshold,but incubation ceased before threshold was reached.

Example 5 Bacterial Conversion of Daidzein to Equol in Food

In an experiment to discover bacteria, or combinations of bacteria, thatcan metabolise daidzein in a reducing environment, samples of adaidzein-enriched soy milk containing approximately 20 mg/L of daidzeinwere inoculated with different bacteria either in pure culture or as acombination of several organisms. The inoculated soy milks wereincubated anaerobically at 37° C. for up to 42 hours Samples werewithdrawn at intervals throughout the time period of the experiment andanalyzed for isoflavone content, in particular the daidzein content.Conversion of daidzein to equol would be accompanied by lowering of thelevel of daidzein in the product over time, with the hydrogenatedproduct, equol, taking its place. No significant changes in isoflavonecontent, outside of the daidzein level, were found in any of theinoculated products, which effectively demonstrates the stability ofisoflavones (including daidzein when suitable metabolizing bacteria areabsent or inactive). The results are shown in Table D. Of sevendifferent innocula studied, four showed no change in daidzein contentduring the full incubation period. Three of the inoculated samplesdemonstrated substantial lowering of the level of daidzein withcorresponding conversion to the hydrogenated compound. The organismseffecting this change were Proprionobacteria freundenreichii, a mixedculture containing: Bifidobacterium lactis, Lactobacillus acidophilus,Lactococcus lactis, Enterococcus faecium, Lactobacillus casei andLactobacillus salivarius; and a mixed culture isolated from equinefeces. Daidzein loss to approximately 50% of the initial level occurredin less than 15 hours with the equine feces mixed culture and took up to25 hours with the other two cultures

TABLE D Conversion of Daidzein During Growth of Various Microorganismsin a Food Base (Incubation at 37° C. under Anaerobic Conditions) Timerequired to metabolize 50% of the daidzein Bacterial species/strainpresent Uninoculated Food medium Not metabolized Propionobacterium acnesNot metabolized Propionobacterium freundenreichii 25 hours Lactobacillusfermentum Not metabolized Mixed culture from equine feces 15 hours Mixedculture containing: Bifidobacterium 25 hours lactis, Lactobacillusacidophilus, Lactococcus lactis, Enterococcus faecium, Lactobacilluscasei and Lactobacillus salivarius Lactobacillus salivarius Notmetabolized Bacterioides vulgatus Not metabolized

Example 6 Bacterial Production of S-Equol in a Food Product

A simple, light broth was prepared containing hydrolyzed plant and milkproteins with salt and sugar. Daidzein, at a level of approximately 2mg/L, was added to the broth. The broth was cooked in a pressure cookerfor about 15 minutes and after cooling to room temperature wasinoculated with a mixed culture of organisms derived from thegastrointestinal tract of a person known to produce equol when consumingsoy milk as part of a regular diet. The broth was held at a temperatureof 37° C. for 24 hours and then analyzed. The live organisms can thenoptionally be destroyed by a method commonly used to deactivateorganisms in a food product. The presence of equol (presumed to beS-equol) derived from the daidzein was confirmed by electrospray massspectrometry of an extract of the broth.

Example 7 Enzymatic Production of S-Equol in a Food Product

A mixed culture of bacteria, containing Bifidobacterium lactis,Lactobacillus acidophilus, Lactococcus lactis, Enterococcus faecium,Lactobacillus casei and Lactobacillus salivarius, is cultured in anutritive tryptone broth anaerobically at 37° C. for from about 24 hoursto 36 hours. The bacteria are separated from the culture broth bycentrifugation at about 10,000 gravities of force, and the cells aresuspended in about 0.9% saline solution, and then are re-centrifuged.The washed, separated cells are used to prepare an extract of activeenzymes using techniques well know to those practicing in the art ofenzymology and biochemistry. The crude enzyme mixture can be used as is,or can be further purified by conventional enzyme preparation techniquesinto a purified enzyme extract.

The purified enzyme mixture is added to a food product that contains 10mg/L daidzein. The composition is incubated for about 2 hours at atemperature of about 30° C. to 40° C. while maintaining mild anaerobicconditions. The enzymes are then inactivated by heating the compositionto about 95° to 100° C., resulting in a food product containing S-equol.

1. An oral pharmaceutical composition consisting essentially of the Senantiomer of equol (S-equol) and a pharmaceutically acceptableadjuvant, carrier or excipient.
 2. The oral pharmaceutical compositionof claim 1 wherein the S-equol has an enantiomeric purity of 90% minimumenantiomeric excess (EE).
 3. The oral pharmaceutical composition ofclaim 2 wherein the S-equol has an enantiomeric purity of 96% minimumEE.
 4. The oral pharmaceutical composition of claim 3 wherein theS-equol has an enantiomeric purity of 98% minimum EE.
 5. The oralpharmaceutical composition according to claim 1 where the S-equol isfurther conjugated at the C-4′ or C-7 position to from a conjugateselected from the group consisting of glucuronide, sulfate, acetate,propionate, glucoside, acetyl-glucoside, malonyl-glucoside, and mixturesthereof.
 6. An oral pharmaceutical composition comprisingenantiomerically pure S-equol as an active agent and a pharmaceuticallyacceptable adjuvant, carrier or excipient.
 7. The oral pharmaceuticalcomposition of claim 1, wherein the composition is formulated as a unitdose.
 8. The oral pharmaceutical composition of claim 7, wherein theunit dose is selected from the group consisting of a tablet, a capsule,a lozenge, and a cachet.
 9. The oral pharmaceutical composition of claim6, wherein the composition is formulated as a unit dose.
 10. The oralpharmaceutical composition of claim 9, wherein the unit dose is selectedfrom the group consisting of a tablet, a capsule, a lozenge, and acachet.
 11. The oral pharmaceutical composition of claim 6 wherein theS-equol has an enantiomeric purity of 90% minimum enantiomeric excess(EE).
 12. The oral pharmaceutical composition of claim 11 wherein theS-equol has an enantiomeric purity of 96% minimum EE.
 13. The oralpharmaceutical composition of claim 12 wherein the S-equol has anenantiomeric purity of 98% minimum EE.